The invention relates to a method for treating biomaterial using at least one electrical field generated by a first voltage pulse which is terminated once the value for an electrical parameter has exceeded or dropped below a preset limit, as well as to a circuit arrangement, in particular for carrying out said method, comprising at least one storage device for electrical charges to generate at least one voltage pulse by selectively discharging the storage device, and at least one control unit for controlling the discharge. The present invention relates in particular to the field of electroporation, electrofusion and electrostimulation of living cells, as well as to all applications in which biomaterial must be exposed to an electrical field.
The introduction of bioactive molecules, e.g., DNA, RNA or proteins, into living cells is an important tool in studying the biological functions of these molecules. One preferred method for introducing foreign molecules into cells here is electroporation, which does, as opposed to chemical methods, not rely on the simultaneous transport of other bioactive molecules. In electroporation, the foreign molecules are taken from a buffer solution adapted to the cells or a cell culture medium and introduced into the cells in a brief flow of current, wherein exposure to the short electrical voltage pulses or resultant electrical field makes the cell membrane permeable to the foreign molecules. The cell suspension is here often in a so-called cuvette, i.e., a narrow flask open at the top, which has two opposing, parallel electrodes in the lateral walls in proximity to its floor, which are used to apply an electrical voltage. Through the briefly arising “pores” in the cell membrane the bioactive molecules initially enter the cytoplasm, where they can already perform the function to be studied, and then, under certain conditions, also the cell nucleus.
Briefly applying a strong electrical field, i.e., a short voltage pulse with a high current density, also makes it possible to fuse cells, cell derivates, sub-cellular particles and/or vesicles. During this so-called electrofusion, the cells are, for example, initially brought into close membrane contact by an inhomogeneous electrical alternating field. The subsequent application of an electrical field pulse then causes the membrane sections to interact, finally resulting in fusion. Industrial equipment comparable to that used for electroporation can here be used for electrofusion. Further, living cells can also be stimulated by electrical fields in such a way as to change their properties.
If, in the process of establishing an electrical field with a field strength of several hundred volts per centimeter in an aqueous solution, the electrical resistance collapses in a very short time, e.g., under 1 μs, thereby causing the current to rise very rapidly and sharply, a so-called lightning discharge can occur. During a lightning discharge, the brief rise in power or heat leads to concomitant physical phenomena, such as lightning, cracking and spraying of the solution on the one hand, and irreversible damaging or killing of the cells on the other hand. Therefore, a lightning discharge generally endangers not only the safety of people and equipment in the vicinity, but also results in a loss of the used biomaterial.
WO 02/086129 A1 discloses a circuit arrangement for introducing bioactive molecules into the cell nucleus of eukaryotic cells by means of an electrical current, or for treating cells, cell derivates, sub-cellular particles and/or vesicles with electrical current, as well as a corresponding method. The circuit arrangement consists of two storage devices for electrical charges, which are each supplied by a high voltage power supply. The storage devices are each connected to a power semiconductor for transmitting the charges present in the storage devices to a cell suspension. The power semiconductors are actuated and switched via a control device. This circuit arrangement further provides for that at least a first voltage pulse can be transmitted to the cell suspension with the capacitor voltage of the storage device by actuating a power semiconductor for a preset time (T1). To further enhance the safety of the user and used samples, it is provided that an overcurrent switching module enables overvoltage deactivation at least for the first voltage pulse, terminating the respective pulse. Therefore, overcurrent deactivation makes it possible to terminate the voltage pulse in a case where preset limits have been exceeded. For example, if the current rises too precipitously while establishing an electrical field, a lightning discharge, and hence cell damage, can be prevented by terminating the voltage pulse. However, depending on the point of termination, the disadvantage of this is that successful treatment is not achieved, e.g., the transfection efficiency is too low. If the voltage pulse is terminated too early, the corresponding reaction batch must be discarded or can only be used to a very limited extent, even though cell viability has been obtained.
There is therefore a need for a method and a circuit arrangement of the kind mentioned at the outset that enable the successful treatment of biomaterial even if the first voltage pulse has been terminated.